The long-term objectives of this proposal are: 1) to identify the steps linking fuel metabolism to cellular Ca2+ homeostasis and insulin release; 2) to determine how these steps are regulated; 3) to understand the defects in the sequence that prevents normal fuel responsiveness in various tumor cells and in Type II diabetics. The specific aims of this project are: 1) to investigate the effects of various fuel and non-fuel secretagogues such as glucose, D-glyceraldehyde, Alpha-ketoisocaproate and acetylcholine on the cytosolic free Ca2+ concentration and intracellular Ca2+ distribution; 2) to determine the effect of various energy state parameters (phosphorylation potential, redox state and pH) on the Ca2+ steady state maintained by the intracellular organelles in permeabilized cells; 3) to study the regulation of inositol 1,4,5-trisphosphate production and action and to assess its role in insulin secretion. The proposed studies will be carried out on insulinoma cells from transplantable tumors and clonal lines as well as with rat pancreatic islets. All of these differ in their secretory characteristics. Ca2+ homeostasis will be studied using several methods (quin 2, Ca2+ mini-electrodes, arsenazo III) that directly monitor free Ca2+ concentration. The result obtained will be correlated with secretion, metabolic, bioenergetic, cytosolic pH and membrane conductance studies in the same cells.